HIV-1 Reverse Transcriptase: Drug Resistance Mechanisms

1. HIV-1 Reverse Transcriptase: Drug Resistance Mechanisms

3. Steps of DNA Polymerization RT RT/DNA RT/DNA/dNTP fingers thumb DNAn PPi dNTP E´/DNAn E´/DNAn+1 E´/DNAn/dNTP E E*/DNAn/dNTP E + DNA Conformational change/ catalysis dNTP binding DNA binding Translocation Sarafianos et al. (1999) Chem. & Biol. 6, R137-R145

4. Nucleoside Analogs and Nonnucleoside RT Inhibitors • Nucleoside analogs (NRTIs) that are used to treat HIV-1 infections all lack the normal 3’ OH and act as chain terminators when incorporated into viral DNA • Nonnucleoside RT inhibitors (NNRTIs) bind to HIV-1 RT near, but not at, the polymerase active site distorting the enzyme and blocking the chemical step of viral DNA synthesis

5. Drug Binding Sites in HIV-1 RT NRTI NNRTI

6. Sites of Drug Resistance Mutations in HIV-1 RT NNRTI resistance mutation sites NRTI resistance mutation sites

8. Nonnucleoside RT Resistance Mechanisms • Mutations that reduce the interactions between RT and the bound drug (Y181C, Y188L). • Mutations that cause steric hindrance with the bound drug (G190A/S). • Mutation that makes it more difficult for the NNRTI to enter the binding pocket (K103N). • Indirect effects (L100I, V106A, V108A).

9. NRTI Resistance • Resistance to nucleoside analogs (NRTIs) implies that the mutant reverse transcriptase (RT) has an enhanced ability to discriminate between the NRTI and the normal dNTPs. • This discrimination can occur if the NRTI is incorporated less efficiently (exclusion) or by enhanced excision of the NRTI after it has been incorporated (ATP-dependent pyrophosphorolysis).

10. NRTI Resistance Mechanisms • Exclusion: M184V, 3TC/FTC resistance; steric hinderance • Exclusion: K65R, Q151M resistance to multiple NRTIs, altered interactions with the 3’OH of the incoming dNTP • Excision: TAMs (T215F/Y and friends) cause AZT resistance by enhanced ATP binding • Fingers insertions: extend TAMs to cause excision of many NRTIs

11. Steric Hindrance in 3TC Resistance

12. AZT Resistance (Excision) • Excision involves pyrophosphorolysis (the reverse of polymerization). • AZT-resistant RTs preferentially excise AZTMP. • AZT-resistance mutations enhance the ability of RT to bind ATP, the in vivo pyrophosphate donor. • AZT is excised easily because the long azido group interferes with translocation: an AZT terminated primer preferentially resides at the N (active) site where it can be excised

13. Polymerization

14. Closed Complex Reduces Excision Stable Complex No Excision b3-b4 Loop

15. AZTMP Excision * * ATP Binding Mutations Unstable Complex Excision AZT MP b3-b4 Loop

16. Y183 primer template P M184 D186 Y115 N D185 AZTMP D110 g D67N b fingers K219E a D113 T215Y K70R M41L ATP

17. An AZT Terminated Primer in the P Site Causes Steric Hindrance with a dNTP in the N site P Site (AZTMP) N Site (dNTP) Active Site

18. AZT Resistance: Q151M • Certain NRTI combination therapies (AZT + ddI + ddC) select Q151M in HIV-1 RT. However, Q151M appears in only 5% of the treated HIV-1 patients. • There is less data (fewer treated patients) but HIV-2 prefers to use the Q151M pathway for AZT resistance • In HIV-1 (but not HIV-2) the Q151Mutation is usually accompanied by additional mutations • Why does HIV-1 predominantly use an ATP-dependent excision pathway (T215Y/F) while HIV-2 predominantly uses an AZT exclusion pathway (Q151M)?

19. AZTTP Inhibition of Polymerization of HIV-1RT, HIV-2 RT, and Their Q151M Mutants

20. HIV-1 RT (WT and Mutants) Are Better at AZTMP Excision than HIV-2 RT (WT and Mutants)

21. ATP Binding sites in HIV-1 RT and HIV-2 RT P P 185 AZTMP N 185 AZTMP N 112 115 ATP 115 215 112 209 113 214 ATP 117 214 116 118 113 116 215 117 41 211 44 5 44 41 4 1 46 46 2 4 3 HIV-1 RT HIV-2 RT

22. HIV-1 and HIV-2 AZT Resistance • Each virus prefers a resistance pathway that is best suited to extend the properties of their respective wild-type RTs. • Viewed in this light, the ability of HIV-1 to develop AZT (and multi-NRTI) resistance using ATP-mediated excision is an unfortunate coincidence based on the existence of a nascent ATP binding site that appears to have no normal function.

23. Delayed Chain Terminators • HIV-1 excision works efficiently because the NRTI remains at the end of the primer strand where it can be excised. • Delayed chain terminators block DNA synthesis several nucleotides after they have been incorporated. • As expected, delayed chain terminators block the excision reaction and inhibit the growth of viruses that replicate using excision proficient RTs.

24. Conclusions • There are a number of distinct mechanisms for NRTI and NNRTI resistance. • Resistance mechanisms evolve as logical extensions of the properties of wild type RT. • Understanding the mechanism(s) of RT resistance makes it possible to develop strategies (and drugs) that counteract these mechanisms .

25. Acknowledgments NCI-Frederick CABM/Rutgers Paul L. Boyer Stefan Sarafianos* Pat Clark Kalyan Das John Julias Eddy Arnold Victor Marquez Tel Aviv Amnon Hizi

27. North-Methanocarbathymidine (N-MCT)

28. N-MCdATP Is a Delayed Chain Terminator T

29. N-MCT Inhibits HIV-1 Viruses that Replicate Using Excision-Proficient RTs in HSV TK+ Cells Relative Infectivity µM North-MCT

30. HIV Infection/Mortality • Over 3 million deaths in 2003; about 7 per minute • 40-50 million infected worldwide • In 2003, about 5 million new infections, mostly in Africa, South and Southeast Asia • About 1 million already infected in the US; about 45,000 new infections and 15,000 deaths in 2003

31. HIV Evolution • HIV replication is error prone: Error rate ca. 1 per genome per replication cycle • The rapid replication of the virus, together with the error rate and high viral load causes the virus to evolve rapidly • The rapid evolution of the virus makes the problems of vaccine development and drug therapy particularly difficult

32. Anti-HIV Drugs and Drug Targets • Target should be essential and conserved; enzymes are better targets (RT, PR, IN) • HIV develops resistance to all drugs • RT has at least two separate drug targets (there may be more); PR and IN have one • Understand resistance; develop drugs that are effective against resistant viruses

33. Anti-HIV Drugs and Drug Resistance • Drugs do not cure an HIV infection • Drugs need to be taken (regularly) for the life of the patient • For drugs to be effective replication must be blocked completely (stop evolution of resistance) • It takes at least three drugs in combination to fully block viral replication/evolution

34. Retroviral Life Cycle

36. Mechanism of K103N Resistance

37. Flexible inhibitor Rigid inhibitor Torsional changes (wiggling) Steric hindrance Reorientation and repositioning (jiggling)

38. Subdomains of the Subunits of HIV-1 RT

39. Two NRTI Drug Resistance Paradigms: Reduced Incorporation and Enhanced Excision

40. M814V Causes Steric Hindrance with 3TCTP

41. Clash Between Incorporated AZT and Active Site Aspartic Acid Prevents Translocation

42. WT Complex N superposed on WT Complex P template primer P site D185 D186 AZTMP D110 N site AZTMP Q151 R72 fingers

43. P site (priming site) Primer dNTP N site (nucleotide binding site) 3’OH a Catalytic carboxylates b g Released as pyrophosphate

44. Y183 primer template P M184 D186 Y115 N D185 AZTMP D110 g D67N b fingers K219E D113 T215Y K70R M41L PPi

45. NRTI Excision * * ATP Binding Mutations Unstable Complex Excision b3-b4 Loop Fingers Mutations

46. Excision/Extension Assay +ATP, dNTPs, NRTITPs

47. HIV-1 RT and HIV-2 RT Sequences HIV-1 (BH10) HIV-2 (ROD) ---PGIRYQYNVLPQGWKGSPAIFQ--- || || | |||||||||||||| ---PGKRYIYKVLPQGWKGSPAIFQ--- 151 ---IYQYMDDLYVGSDLEIGQHRTKIEELRQHLLRWGLTTPDKKHQKEPP--— | |||||• ••|| | • |• | | •||| | ||•|| ---IIQYMDDILIASDRTDLEHDRVVLQLKELLNGLGFSTPDEKFQKDPP--- 185215 219

48. ATP-Mediated AZTMP Excision/Extension Deblock and Extend 100 uM each dNTP vary ATP

49. PPi-Mediated AZTMP Excision/Extension Deblock and extend 100 uM each dNTP vary PPi

50. Q151 in HIV-1 and HIV-2 RT 74 115 151 73 HIV-1 /DNA/dNTP HIV-1 116 HIV-2